The N-methyl-D-aspartate receptor (NMDAR) plays a part in the induction of apoptotic cell death following cerebral insults. The molecular components of the NMDAR have recently been elucidated. The receptor is a multi-subunit protein composed of individual subunits termed NR1, NR2, and NR3. Furthermore, the NR2 subunit family is further subdivided into NR2A, NR2B, NR2C and NR2D subtypes. Typically, the NMDAR is composed of a combination of these subunits, with NR1 always present along with at least one NR2 subunit and, less often, an NR3 subunit (Das, S., et al., (1998) Nature, 393:377-381; Chatterton, J. E., et al., (2002) Nature, 415:793-798). Each of the subunits confers a slightly different pharmacological profile onto the NMDAR. The existence of multiple subunit subtypes and the molecular heterogeneity of the fully assembled NMDAR paves the way for rational drug design strategies and the development of subunit specific medications for glutamate excitotoxicity (for a review see, Kemp, J. A. and McKeman, R. M., (2002) Nature Neuroscience Supp., 5:1039-1042). However, although such compounds may be subunit selective, some antagonists may affect NMDAR gating and therefore cause unwanted side effects in additional to any beneficial effects that they may have.
To date, clinical trials in stroke and brain injury testing the efficacy of therapeutics that function by blocking NMDA receptors have not been successful (Kemp, J. A., et al., Handbook of Experimental Pharmacology, Vol. 141 (eds. Jonas, P. & Monyer, H.), 495-527 (Springer, Berlin, 1999); Lees, K. R., et al., (2000) Lancet, 355:1949-1954; Sacco, R. L., et al., (2001) JAMA, 285: 1719-1728). Since a properly functioning NMDAR is critical in, among other things, refining synaptic connections, long term potentiation, learning and memory, blockers of the receptor may produce unwanted side-effects. Molecularly, since these drugs block calcium influx, they therefore also inhibit the initiation of a multitude of beneficial cell signaling pathways in addition to the ERK/MPK pathway involved in triggering apoptosis. Clinically multiple different side effects have been observed with NMDAR blockers, including hallucinations, increases in blood pressure, memory disruptions, catatonia and the development of schizophrenia like symptoms.
Subunit specific antagonists alleviate some, but not all, of the above described problems. Given that these antagonists are specific to a particular subunit, side effects are somewhat minimized since only NMDARs carrying that specific subunit are blocked. For example, compounds such as ifenprodil, CP-101606, Ro 25-6981 and Ro 63-1908 are all NR2B specific, but they all function by altering the gating of the NMDAR (Williams, K., (1993) Mol. Pharmacol., 44: 851-859; Kemp, J. A., et al., Handbook of Experimental Pharmacology, Vol. 141 (eds. Jonas, P. & Monyer, H.), 495-527 (Springer, Berlin, 1999); Gill, R., et al., (2002) J. Pharmacol. Exp. Ther., 302: 940-948). Although these antagonists are in some ways superior to non-specific NMDAR blockers, they still elicit unwanted side effects by virtue of blocking calcium influx through the receptor and, therefore, blocking all cell signaling that is independent of the ERK/MAPK cascade.
In addition to antagonists directed at the NMDA receptor, blockers acting at sites upstream to the ERK/MAPK cascade as well as blockers directed at components of the ERK/MAPK cascade are being developed. However, all of these antagonists suffer from the same disadvantage in the sense that all completely block their target resulting in severe side effects that seriously undermine their clinical utility (Ikonomidou C, et al. (1999) Science, January 1;283(5398):70-4).
Glutamate promotes the activation of the NMDAR, which initiates multiple cellular events, including an increase in postsynaptic calcium (Ghosh, A., et al., (1995) Science 268, 239-47) and the activation of the Extracellular Signal—Regulated Kinase (ERK1/2 or ERK/MAPK) pathway (Sweatt, J. D. (2001), J Neurochem 76, 1-10). These events activate multiple transcription factors (Platenik, J., et al., (2000), Life Sci 67, 335-64, West, A. E. et al. (2001), Proc Natl Acad Sci U S A 98, 11024-31) that play a central role in the synaptic plasticity underlying refinement of neuronal connections during development, long-term potentiation, learning, and memory (Platenik, J., et al., (2000), Life Sci 67, 335-64, Abel, T. et al., (2001), Curr Opin Neurobiol, 11, 180-7 (2001)). However, excess concentrations of glutamate and the resultant repetitive stimulation of NMDAR receptor result in toxic concentrations of postsynaptic calcium and the initiation of a lethal cellular cascade that culminates in cell death. Excessive release of glutamate activates NMDA receptors which in turn activates the ERK/MAPK pathway and multiple downstream transcription factors, including Fos, Jun, and Egr, which are known in the art to play a central role in glutamate induced cell death. Thus, the NMDAR-dependent ERK/MAPK pathway might be one of the first elements in a cascade of events leading to neuronal cell death.
Multiple pathological conditions can result in a massive release of glutamate, including cerebral infarcts, head traumas, anoxic insults or seizures of various etiologies, including epilepsy. Additionally, an increase in the release of glutamate is associated with complex neurodegenerative disorders such as Parkinson's disease and Huntington's disease. The role of NMDA receptors in Parkinson's symptoms is well documented (Nash J E. Brotchie J M. (2002) Movement Disorders, 17(3):455-466; Li D. A., Lipton S A., (2001) Drugs & Aging, 18(10):717-724). Cerebral infarcts alone are a major cause of morbidity and mortality with 750,000 cases per year in the United States and 500,000 in Europe. The only currently available treatment for cerebral infarcts is tissue plasminogen activator (TPA), which is only effective if administrated within a few hours of the cerebral insult. Furthermore, while TPA may be effective in minimizing cellular damage by acting on the blood clot causing the damage, it does not in anyway prevent the initiation of the ERK/MAPK signaling cascade which triggers apoptosis. Anoxic insults resulting from cardiac arrest incapacitate as many as 1.5 million people a year and only 10% of those individuals are able to resume active lives following the event. In addition, as many as 30 to 50% of existing epileptic patients require hospitalization due to incapacitating temporal lobe seizures.
Given the devastating consequences of glutamate toxicity, therapeutics capable of preventing glutamate induced cell death, and assays for identifying the same, are needed.